Abstract text: Plant cell wall–derived oligosaccharides generated during pathogen attack function as damage-associated molecular patterns (DAMPs) that trigger immune responses. Characterizing glycan-based DAMPs released in vivo during pathogen colonization by the activity of cell wall-degrading enzymes (CWDEs) remains challenging. To overcome these limitations, we have developed a controlled in vitro strategy for novel DAMP identification. We have extracted and purified polysaccharides from plant cell walls to remove pre-existing oligosaccharides, and then we have selectively hydrolyzed them using plant pathogens or CWDEs. This ensures that released oligosaccharides are derived exclusively from CWDE activity. In parallel, a semi-in vivo approach using dried and ground leaf material was implemented to preserve the complexity of native cell walls, which are later subjected to microbial degradation. Using these methodologies, we have identified novel oligosaccharides (DAMPs) and studied the receptors involved in their perception. This approach was further used to functionally characterize the activity of pathogen CWDEs by comparing fungal wild-type strains and CWDE-deficient mutants. This work provides a reproducible framework for discovering novel plant cell wall-derived DAMPs and examining pathogen CWDE activities and offers new insights into plant-pathogen interactions and innate immune activation. This knowledge can be used to design novel, sustainable crop protection solutions.